The present device relates to methods for analyzing whole blood using immunoassay and other assay devices in which a fluid is analyzed by contacting the fluid with a porous support on which are immobilized agents that selectively bind the analyte.
Devices that isolate analytes from solution by binding them to a solid porous support eliminate the need for precipitation and centrifugation, or filtration, and have made it possible to perform many diagnostic tests at home or in clinical settings where there are no laboratory facilities. However, the extra-laboratory application of these devices has not been fully realized because the interpretation of these "blood tests," like those of several other diagnostic methodologies, requires that they be carried out on blood serum or plasma, either of which must be separated from the cellular components of whole blood by centrifugation prior to the analysis.
The interpretation of porous support assays depends on the observance of color at the locus of the bound analyte where a chromogenic reaction occurs. If hemoglobin, the highly colored component of erythrocytes, stains the same field, an accurate observation is difficult or impossible. Not only hemoglobin, but also colorless insoluble components of blood can impair the assay. In addition, the cellular material of intact erythrocytes or their ruptured "ghosts" can reduce sensitivity of the test by blanketing analyte binding sites on membrane surfaces. Blood cells or cellular debris can alter test performance in yet another way by clogging membrane pores and restricting sample flow.
Some procedures of this type (e.g., Reflotron, Boehringer Mannheim Diagnostics, Indianapolis, Ind.) have been adapted to whole blood analysis by providing a glass fiber prefilter which separates erythrocytes from whole blood by adhesion of the cells to the glass fibers. To achieve efficient erythrocyte separation and reliable assay results using a prefilter of this type, the blood must be tested within a few minutes after collection or else treated with an anticoagulant. Once the coagulation process has begun, contact with the large surface area of the glass fiber mat will accelerate fibrin clot formation, and the flow through the prefilter and porous membrane as well will be restricted. The glass fibers retain only intact erythrocytes; they will not retain hemoglobin from lysed red blood cells. There is some degree of hemolysis in all collected blood; some specimens, notably those of cord blood from infants, are virtually impossible to obtain in an unhemolyzed state, even with a careful collection. Thus, even with the use of a prefilter, hemoglobin staining from whole blood samples will contaminate the observed analytical field.
The extra-laboratory potential of many diagnostic tests could be fully realized if the separating properties of porous membrane assay devices could be applied to exclude cellular components of blood together with soluble hemoglobin in the process of isolating the analyte.
It is therefore an object of the invention to provide such a whole blood assay system and to define the physical and operating parameters of these assay systems which best achieve the desired result.